Controllable Transgene Expression Systems in Plants
One of the major problems in plant biotechnology is the achievement of a reliable control over transgene expression. Tight control over gene expression in plants is essential if a downstream product of transgene expression is growth inhibitory or toxic, like for example, biodegradable plastics (Nawrath, Poirier & Somerville, 1994, Proc. Natl. Acad. Sci., 91, 12760-12764; John & Keller, 1996, Proc. Natl. Acad. Sci., 93, 12768-12773; U.S. Pat. No. 6,103,956; U.S. Pat. No. 5,650,555) or protein toxins (U.S. Pat. No. 6,140,075).
Existing technologies for controlling gene expression in multicellular organisms, especially in plants, are usually based on tissue-specific or inducible promoters and practically all of them suffer from a basal expression activity even when uninduced, i.e. they are “leaky”. Tissue-specific promoters (U.S. Pat. No. 0,595,5361; WO09828431) represent a powerful tool but their use is restricted to very specific areas of applications, e.g. for producing sterile plants (WO9839462) or expressing genes of interest in seeds (WO00068388; U.S. Pat. No. 0,560,8152). Inducible promoters can be divided into two categories according to their induction conditions: those induced by abiotic factors (temperature, light, chemical substances) and those that can be induced by biotic factors, for example, pathogen or pest attack. Examples of the first category are heat-inducible (U.S. Pat. No. 0,518,7287) and cold-inducible (U.S. Pat. No. 0,584,7102) promoters, a copper-inducible system (Mett et al., 1993, Proc. Natl. Acad. Sci., 90, 4567-4571), steroid-inducible systems (Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; U.S. Pat. No. 0,606,3985), an ethanol-inducible system (Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334), and a tetracycline-inducible system (Weinmann et al., 1994, Plant J., 5, 559-569). One of the latest developments in the area of chemically inducible systems for plants is a chimaeric promoter that can be switched on by the glucocorticoid dexamethasone and switched off by tetracycline (Bohner et al., 1999, Plant J., 19, 87-95). For a review on chemically inducible systems see: Zuo & Chua, (2000, Current Opin. Biotechnol., 11, 146-151). Other examples of inducible promoters are promoters which control the expression of pathogenesis-related (PR) genes in plants. These promoters can be induced by treatment of a plant with salicylic acid, an important component of plant signaling pathways in response to pathogen attack, or other chemical compounds (benzo-1,2,3-thiadiazole or isonicotinic acid) which are capable of triggering PR gene expression (U.S. Pat. No. 5,942,662).
There are reports of controllable transgene expression systems using viral RNA/RNA polymerase provided by viral infection (for example, see U.S. Pat. No. 6,093,554; U.S. Pat. No. 5,919,705). In these systems, a recombinant plant DNA sequence includes the nucleotide sequences from the viral genome recognized by viral RNA/RNA polymerase. The effectiveness of these systems is limited because of the low ability of viral polymerases to provide functions in trans, and their inability to control processes other than RNA amplification.
Another way is to trigger a process of interest in a transgenic plant is by using a genetically-modified virus which provides a heterologous nucleic acid encoding a switch for a biochemical process in a genetically-modified plant (WO02068664).
The systems described above are of significant interest as opportunities of obtaining desired patterns of transgene expression, but they do not allow tight control over the expression patterns, as the inducing agents (copper) or their analogs (brassinosteroids in case of steroid-controllable system) can be present in plant tissues at levels sufficient to cause residual expression. Additionally, the use of antibiotics and steroids as chemical inducers is not desirable or economically unfeasible for large-scale applications. When using promoters of PR genes or viral RNA/RNA polymerases as control means for transgenes, the requirements of tight control over transgene expression are also not fulfilled, as casual pathogen infection or stress can cause expression. Tissue- or organ-specific promoters are restricted to very narrow areas of application, since they confine expression to a specific organ or stage of plant development, but do not allow the transgene to be switched on at will. Recombinant viral switches as described in WO02068664 address all these problems, but do not guarantee tight environmental safety requirements, as the heterologous nucleic acid in the viral vector can recombine.
There is an abundant literature including patent applications which describe the design of virus resistant plants by the expression of viral genes or mutated forms of viral RNA (e.g. U.S. Pat. No. 5,792,926; U.S. Pat. No. 6,040,496). It is also worth mentioning that an environmental risk is associated with the use of such plants due to the possibility of forming novel viruses by recombination between the challenging virus and transgenic viral RNA or DNA (Adair & Kearney, 2000, Arch. Virol, 145, 1867-1883).
Protein-protein interactions as switch of gene expression represent an interesting choice due to several advantages over the existing controllable systems. The system built on protein-protein interactions may be rendered highly specific, as the function of interest is a result of a highly specific protein-protein or protein-nucleic acid interaction, which is characterized by near zero-level expression in the uninduced state and absence of non-specific leakiness in cases when the activation of gene of interest is dependent on nucleic acid (DNA or RNA) rearrangement, said nucleic acid is encoding and/or controlling said gene. This is in contrast to existing systems such as switches based on small molecules that are inherently less specific and invariably show a certain degree of leakiness.
However, all systems described above suffer from at least one of the following two severe problems: (i) the lack of tightly controlled regulation, e.g. “leakiness” of the system; (ii) the induction of a cellular process is usually restricted to a limited number of cells within a multicellular organism, especially in the case of a tightly regulated and highly specific regulatory system built on e.g. protein-protein or protein-nucleic acid interactions. Usually the tighter the regulation, the smaller is the number of cells in a multi-cellular organism that can be affected by an externally applied signal for inducing the cellular process of interest. An example of this problem are the publications of Hooykaas and colleagues (2000, Science, 290, 979-982; WO0189283) describing a specifically triggered process of expressing a gene of interest conferring antibiotic resistance to plant cells. This process is specifically triggered by the externally delivered enzyme Cre recombinase. However, the efficiency of applying the external trigger is low, as Cre recombinase can be delivered only to a small fraction of the target cells. As a result, cells expressing the resistance gene can be detected in tissue culture due to their selectable phenotype caused by antibiotic resistance. However, this method is limited to issue culture and cannot be applied to whole multi-cellular organisms like higher plants.
Therefore, it is an object of the present invention to provide an environmentally safe method of controlling a cellular process of interest in a multi-cellular organism, notably a higher plant, whereby the cellular process can be efficiently and selectively activated in said multi-cellular organism or a part thereof. It is another object of the invention to provide a method for producing a product in a genetically-modified multi-cellular organism, notably a higher plant, wherein the production of the product may be selectively switched on after the multi-cellular organism has grown to a desired stage.